Microbially-expressed thermotolerant phytase for animal feed

ABSTRACT

The invention provides methods for making and using thermotolerant phytases, e.g., a method of using a thermotolerant phytase in feed and food processing and feed or food products comprising a thermotolerant phytase.

RELATED APPLICATION

This application claims priority to Application No. 60/344,523, filedDec. 28, 2001, which is incorporated by reference herein.

FIELD OF THE INVENTION

The present invention generally relates to the field of molecularbiology, and more specifically, to the use of a thermotolerant phytase.

BACKGROUND OF THE INVENTION

Phytases (myo-inositol hexakisphosphate phosphohydrolase: EC 3.1.3.8)are enzymes that hydrolyze phytate (myo-inositol hexakisphosphate) tomyo-inositol and inorganic phosphate. The enzymes are known to bevaluable feed additives. At the close of the twentieth century, annualsales of phytase as an animal feed additive were estimated to exceed$100 million and were growing.

Poultry and pig diets are currently based primarily on cereals, legumes,and oilseed products. About two-thirds of phosphorus (P) present inthese feedstuffs occur as phytates, the salts of phytic acid(myo-inositol hexakisphosphate, InsP6) (Jongbloed et al., 1993). Phytatephosphorus in plants is a mixed calcium-magnesium-potassium salt ofphytic acid that is present as chelate and its solubility is very low(Pallauf and Rimbach, 1997). Phosphorus in this form is poorlydigestible/available for monogastric animals such as human, swine, andpoultry.

For the utilization of phytate phosphorus and minerals and traceelements bound in phytic acid complexes, hydrolysis of the ester-typebonded phosphate groups of phytic acid by phytase is necessary (Rimbachet al., 1994). Phytases belong to a special group of phosphatases whichare capable of hydrolyzing phytate to a series of lower phosphate estersof myo-inositol and phosphate. Two types of phytases are known:3-phytase and 6-phytase, indicating the initial attack of thesusceptible phosphate ester bond. Although monogastric animals lacksufficient phytase to effectively utilize phytate phosphorous, manyfungi, bacteria and yeasts produce phytase that can be used tosupplement animal rations.

The beneficial effects of supplementary phytases on phosphorusdigestibility and animal performance have been well documented (Mroz etal., 1994; Komegay et al., 1996; Rao et al., 1999; Ravindran et al.,1999). However, most of these studies have been performed on an ad hocbasis with often only superficial information of the enzymes provided asmarketing strategies by the manufacturers. The efficacy of any enzymepreparation depends not only on the type, inclusion rate and level ofactivity present, but also on the ability of the enzyme to maintain itsactivity in the different conditions encountered through thegastrointestinal tract and the conditions used for the pre-treatment ofa food or feed formulation.

Although numerous phytases are available for use as a supplement, manyof the enzymes have certain disadvantages. For example, many of thecurrently used phytases lose activity during feed pelleting processesdue to heat treatment. Additionally, many of the currently used phytasesare not adequate in diets containing low levels of supplemental calciumphosphate.

Thus, what is needed is a phytase with improved properties for animalfeed and food processing.

SUMMARY OF THE INVENTION

Accordingly, the invention provides methods of preparing and using anucleic acid molecule (polynucleotide) which encodes a thermotolerantphytase, i.e., a thermotolerant phytase which retains at least 40%activity after 30 minutes at about 60° C., and which has a high specificactivity, i.e., at least about 200 U/mg at 37° C. and at acid pH, e.g.,pH 4.5. In one embodiment, the invention provides a method to prepare athermotolerant phytase. The method comprises expressing in a microbialhost cell an expression cassette comprising a promoter operably linkedto a nucleic acid molecule encoding a thermotolerant phytase whichretains at least 40% activity after 30 minutes at 60° C. and has aspecific activity of greater than 200 U/mg at pH 4.5 and 37° C. Themicrobial host cell may be a prokaryotic cell, such as a bacterial cell(e.g., Escherichia, Pseudomonas, Lactobacillus, and Bacillus), yeast(e.g., Saccharomyces, Schizosaccharomyces, Pichia or Hansuela) or fungal(e.g., Aspergillus or Trichoderma) cell. In one preferred embodiment,the microbial cell which is employed to prepare the recombinantthermotolerant phytase yields a glycosylated form of the recombinantthermotolerant phytase.

It is preferred that the polynucleotide that encodes the thermotolerantphytase (the first polynucleotide) is operably linked to at least oneregulatory sequence, such as a promoter, an enhancer, an intron, atermination sequence, or any combination thereof, and, optionally, to asecond polynucleotide encoding a signal sequence, which directs theenzyme encoded by the first polynucleotide to a particular cellularlocation e.g., an extracellular location. Promoters can be constitutivepromoters or inducible (conditional) promoters. As described herein,mutagenesis of a parent (bacterial) polynucleotide encoding a phytasewas employed to prepare variant (synthetic) DNAs encoding a phytasehaving improved properties relative to the phytase encoded by the parentpolynucleotide. In one embodiment, the mutations in a number of thevariant DNAs were combined to prepare a synthetic polynucleotideencoding a phytase with enhanced thermotolerance and gastric stabilityand having a similar or a higher specific activity relative to thephytase encoded by the parent polynucleotide. A parent polynucleotidemay be obtained from any source including plant, bacterial or fungalnucleic acid, and any method may be employed to prepare a syntheticpolynucleotide of the invention from a selected parent polynucleotide,e.g., combinatorial mutagenesis, recursive mutagenesis and/or DNAshuffling.

Thus, in one embodiment of the invention, the thermotolerant phytase hasone or more amino acid substitutions relative to a corresponding(reference) phytase, which substitutions are associated with theretention of activity at temperatures equal to or greater than 60° C.Preferably, the thermotolerant phytase has at least 40% activity atabout 60° C. for 30 minutes, more preferably at least 40% activity atabout 65° C. for 30 minutes, even more preferably at least 35% activityat 70° C. for 30 minutes, and which has a specific activity of at least400 U/mg, more preferably at least 600 U/mg, and even more preferably atleast 800 U/mg, at 37° C. and at acid pH, e.g., less than pH 5.0 andmore preferably less than pH 4.0 and greater than pH 1.5. An exemplarythermotolerant phytase of the invention is provided in SEQ ID NO: 1.

Also provided by the invention are vectors which comprise the expressioncassette or polynucleotide of the invention and transformed microbialcells comprising the polynucleotide, expression cassette or vector ofthe invention. A vector of the invention can encode more than onepolypeptide including more than one thermotolerant phytase or may encodea fusion polypeptide comprising the thermotolerant phytase of theinvention, and a transformed microbial cell may comprise one or morevectors of the invention. The transformed cells of the invention areuseful for preparing the recombinant thermotolerant phytase of theinvention. Accordingly, the invention provides thermotolerant phytaseisolated from the transformed microbial cells of the invention, as wellas synthetically prepared enzyme.

Further provided by the invention are methods for formulation ofthermotolerant phytases, phytase formulations or formulated enzymemixtures. The recombinant thermotolerant phytase or formulations thereofmay be added as a supplement to food or animal feed or to components offood and feed prior to, during, or after food or feed processing.Preferably, the recombinant thermotolerant phytase of the invention isadded to a mixture of feed components prior to and/or during heat (e.g.,steam) conditioning in a pellet mill. Thus, the invention includesmethods of making and using a thermotolerant phytase.

Further, as a phytase of the invention is capable of surviving the heatconditioning step encountered in a commercial pellet mill during feedformulation, the invention provides a method of making animal feed,e.g., hard granular feed pellets comprising the thermotolerant phytase.To make feed, the formulated phytase may be mixed with feed components,the mixture steam conditioned in a pellet mill such that at least 50% ofthe pre-heat treated enzymatic activity is retained, and the feedextruded through a pellet dye. The phytase may thus be used as asupplement in animal feed by itself, in addition with vitamins,minerals, other feed enzymes, agricultural co-products (e.g., wheatmiddlings or corn gluten meal), or in a combination therewith. Theenzyme may also be added to mash diets, i.e., diets that have not beenthrough a pelletizer.

Because the currently available commercial phytase enzymes are notthermotolerant, they are often applied post pelleting, generally viaspraying an enzyme solution onto pelleted feed. Some of the problemsassociated with spraying methods are that only a low percentage of thepellets are contacted with enzyme, the enzyme is only present on thesurface of the coated pellets, and feed mills need to invest in andoperate complex spraying machinery. In contrast, the thermotolerantphytase of the invention, which has an 8-fold higher specific activitythan other commercially available enzymes, may be added prior topelleting, thereby facilitating production of a feed with an improveddistribution of the enzyme. Moreover, feed comprising the thermotolerantphytase of the invention may have a longer shelf life than feed sprayedwith phytase, as the spraying process introduces moisture which cansupport fungal and bacterial growth during storage. Further, the higherspecific activity of the thermotolerant phytase of the invention allowsfeed manufacturers to use significantly lower phosphate levels in feed.For example, it is currently recommended that diets supplemented withthe available commercial phytases use a basal level of 0.45% inorganicphosphate. The thermotolerant phytase of the invention may be used witha lower phosphate supplementation, e.g., about 0.225% in poultry diets.

The invention thus provides a method of preparing animal feed comprisingproviding a mixture comprising one or more feed components and apreparation comprising the thermotolerant phytase of the invention, andtreating the mixture under appropriate conditions of temperature andmoisture so as to hydrolyze phytic acid which is present in the mixture.Also provided is animal feed prepared by such a method. Further providedis a method of preparing a thermotolerant phytase containing compositionfor feed formulation comprising combining a liquid solution comprisingthe thermotolerant phytase of the invention and meal flour, e.g., soymeal flour, to yield a mixture; and lyophilizing the mixture to yield alyophilized composition.

The invention further provides a method in which a mixture comprisinganimal feed components and a preparation comprising the thermotolerantphytase of the invention is treated with heat so as to yield aheat-treated animal feed mixture. Heat-treated animal feed prepared bythe method is also provided. The phytase preparation may be a liquid ora solid preparation, and preferably comprises less than about 1%inorganic phosphate. In one embodiment, a liquid solution comprising thethermotolerant phytase of the invention is combined with soy meal flourto yield a mixture and the mixture is then lyophilized. The mixture,which preferably comprises less than 0.45% inorganic phosphate, may alsocomprise at least one vitamin, mineral, an enzyme other than athermotolerant phytase, an organic acid, a probiotic product, anessential oil or a grain processing co-product. The heat-treated feedmay be further processed, for example, by extruding the heat-treatedfeed through a pellet mill to yield pelletized animal feed. Alsoprovided is an animal feed composition comprising the thermotolerantphytase of the invention, and an enzyme feed additive or a food additivecomprising such a thermotolerant phytase.

Also provided is a method of decreasing the feed conversion ratio andincreasing the weight gain of an animal comprising feeding to an animala feed comprising the thermotolerant phytase. Further provided is amethod of minimizing dietary requirements of phosphorus, e.g.,inorganic-phosphorous, in an animal. The method comprises feeding to ananimal a feed comprising the thermotolerant phytase of the invention inan amount effective to increase the bioavailability of phosphorus,preferably the bioavailability of inorganic phosphorous, in the feed tothe animal. Also provided is a method of enhancing the utilization ofphosphorus present in feed for an animal, which method comprises feedingto the animal a feed comprising the thermotolerant phytase of theinvention in an amount effective to increase the bioavailability ofphosphorus in the feed to the animal.

In addition, the invention provides a method of decreasing the phosphatelevels in excreta from an animal comprising feeding to the animal a feedcomprising less than 0.45% inorganic phosphorus and the thermotolerantphytase of the invention in an amount effective to lower levels ofphosphate in the excreta of the animal.

The invention provides a method of improving the nutritive value ofanimal feed or human food. The method comprises adding thethermotolerant phytase of the invention during the preparation of animalfeed or human food. Also provided is a method of preparing human foodcomprising providing a mixture of a food component and a preparationcomprising the thermotolerant phytase of the invention; and treating themixture under appropriate conditions of temperature and moisture tofacilitate the hydrolysis of phytic acid present in the mixture.

Animals within the scope of the invention include polygastric animals,e.g., calves, as well as monogastric animals such as swine, poultry(e.g., chickens, turkeys, geese, ducks, pheasant, grouse, quail andostrich), equine, ovine, caprine, canine and feline, as well as fish andcrustaceans. The levels of phytase in feed or food are preferably about50 to 5000 U/kg, more preferably 100 to 1200 U/kg, or 300 to 1200 U/kg.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates the residual phytase activity after heating whole E.coli cells having wild-type or mutant phytase genes for one hour atvarious temperatures. Mutants prepared by Gene Site SaturationMutagenesis (“GSSM”) developed by Diversa Corporation and havingspecific amino acid substitutions are designated 1x-4x, 4x11, 5x-7x(which have one mutation or up to eight individual amino acidsubstitutions) and NOV9X (which has all eight amino acid substitutions,see SEQ ID NO:1).

FIG. 2 shows wild-type and mutant NOV9X(SEQ ID NO:1) phytase activity inaqueous solution at elevated temperatures versus time.

FIG. 3A illustrates the residual phytase activity of wild-type andmutant NOV9X enzyme after heating for 30 minutes in aqueous solution atvarious temperatures.

FIG. 3B illustrates the residual phytase activity of wild-type andmutant NOV9X enzyme after heating at 100° C. in aqueous solution for upto 8 minutes.

FIG. 4 shows the gastric stability half-lives and residual activityfollowing 5 minutes at 95° C. in aqueous solution of mutant NOV9Xderived from expression in various hosts.

FIG. 5 illustrates feed conversion efficiencies obtained in chickens feddiets containing various levels of supplemental mutant NOV9X enzyme orNatuphos (an Aspergillus phytase enzyme) with different inorganicphosphate supplementations

FIG. 6 shows weight gain data for chicken fed pelleted feed containingNOV9X enzyme obtained from various recombinant hosts or Natuphos.

FIG. 7 shows an LS means plot of the benefit of Nov9X phytase andNatuhos from cumulative reivw of data from nine trials. FCReff LSmeansis the average benefit in points of FCR on use of the enzyme. Coli andNat were significantly different p=0.0291 when the modelFCReff=intercept, Enzyme (coli or natuphos), Dose, control FCR, maize %,dietary metabolisable energy content, diet calcium content, fat content(animal and vegetable), stocking density was employed. R-square=0.92,model p=<0.0001.

FIG. 8 shows an LS means plot of benefit of Zymetrics Nov9x phytase(Coli) and Natuphos (Nat) from cumulative review of data from 9 trials.Gneff LSmeans is the average benefit in grams of gain on use of theenzyme. Coli (benefit 109 grams) and Nat (benefit 63 grams) weresignificantly different from zero and from one another (p=0.0029) whenthe model gain effect=intercept, Enzyme (Coli or Natuphos), Dose, maize%, wheat %, dietary metabolisable energy content, diet total phosphoruscontent and lighting regimen was employed. R-square=0.94, modelp=<0.0001.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

A “microbial” host cell as used herein refers to a bacterium, yeast andfungus.

“Altered levels” refers to the level of expression in transformed ortransgenic cells or organisms that differs from that of normal oruntransformed cells or organisms.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of protein from anendogenous gene or a transgene.

“Chimeric” is used to indicate that a DNA sequence, such as a vector ora gene, is comprised of more than one DNA sequences of distinct originwhich are fused together by recombinant DNA techniques resulting in aDNA sequence, which does not occur naturally. The term “chimeric gene”refers to any gene that contains 1) DNA sequences, including regulatoryand coding sequences, that are not found together in nature, or 2)sequences encoding parts of proteins not naturally adjoined, or 3) partsof promoters that are not naturally adjoined. Accordingly, a chimericgene may comprise regulatory sequences and coding sequences that arederived from different sources, or comprise regulatory sequences andcoding sequences derived from the same source, but arranged in a mannerdifferent from that found in nature.

“Chromosomally-integrated” refers to the integration of a foreign geneor DNA construct into the host DNA by covalent bonds. Where genes arenot “chromosomally integrated” they may be “transiently expressed.”Transient expression of a gene refers to the expression of a gene thatis not integrated into the host chromosome but functions independently,either as part of an autonomously replicating plasmid or expressioncassette, for example, or as part of another biological system such as avirus.

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that provideresistance to antibiotics such as tetracycline, hygromycin orampicillin, or other means for selection of transformed cells.

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence and excludes the non-coding sequences whichare 5′ and 3′ to the coding sequence. It may constitute an“uninterrupted coding sequence”, i.e., lacking an intron, such as in acDNA or it may include one or more introns bounded by appropriate splicejunctions. An “intron” is a sequence of RNA which is contained in theprimary transcript but which is removed through cleavage and re-ligationof the RNA within the cell to create the mature mRNA that can betranslated into a protein.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

The term “contacting” may include any method known or described forintroducing a nucleic acid segment into a cell.

“Expression” refers to the transcription and/or translation of anendogenous gene or a transgene in a host cell. For example, in the caseof antisense constructs, expression may refer to the transcription ofthe antisense DNA only. In addition, expression refers to thetranscription and stable accumulation of sense (mRNA) or functional RNA.Expression may also refer to the production of protein.

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The expression cassettecomprising the nucleotide sequence of interest may be chimeric, meaningthat at least one of its components is heterologous with respect to atleast one of its other components. The expression cassette may also beone which is naturally occurring but has been obtained in a recombinantform useful for heterologous expression. The expression of thenucleotide sequence in the expression cassette may be under the controlof a constitutive promoter or of an inducible promoter which initiatestranscription only when the host cell is exposed to some particularexternal stimulus.

The “expression pattern” of a promoter (with or without enhancer) is thepattern of expression levels. Expression patterns of a set of promotersare said to be complementary when the expression pattern of one promotershows little overlap with the expression pattern of the other promoter.The level of expression of a promoter can be determined by measuring the‘steady state’ concentration of a standard transcribed reporter mRNA.This measurement is indirect since the concentration of the reportermRNA is dependent not only on its synthesis rate, but also on the ratewith which the mRNA is degraded. Therefore the steady state level is theproduct of synthesis rates and degradation rates.

The rate of degradation can however be considered to proceed at a fixedrate when the transcribed sequences are identical, and thus this valuecan serve as a measure of synthesis rates. When promoters are comparedin this way techniques available to those skilled in the art arehybridization S1-RNAse analysis. Northern blots and competitive RT-PCR.This list of techniques in no way represents all available techniques,but rather describes commonly used procedures used to analyzetranscription activity and expression levels of mRNA.

The analysis of transcription start points in practically all promotershas revealed that there is usually no single base at which transcriptionstarts, but rather a more or less clustered set of initiation sites,each of which accounts for some start points of the mRNA. Since thisdistribution varies from promoter to promoter the sequences of thereporter mRNA in each of the populations would differ from each other.Since each mRNA species is more or less prone to degradation, no singledegradation rate can be expected for different reporter mRNAs. It hasbeen shown for various eukaryotic promoter sequences that the sequencesurrounding the initiation site (‘initiator’) plays an important role indetermining the level of RNA expression directed by that specificpromoter. This includes also part of the transcribed sequences. Thedirect fusion of promoter to reporter sequences would therefore lead tosuboptimal levels of transcription.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency(Turner et al., 1995).

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, gene refers to a nucleic acid fragment that expresses mRNA,or specific protein, including regulatory sequences. Genes also includenonexpressed DNA segments that, for example, form recognition sequencesfor other proteins. Genes can be obtained from a variety of sources,including cloning from a source of interest or synthesizing from knownor predicted sequence information, and may include sequences designed tohave desired parameters.

“Genetically stable” and “heritable” refer to chromosomally-integratedgenetic elements that are stably maintained in the host cell and stablyinherited by progeny through successive generations.

“Genome” refers to the complete genetic material of an organism.

The terms “heterologous DNA sequence,” “exogenous DNA segment” or“heterologous polynucleic acid,” as used herein, each refer to asequence that originates from a source foreign to the particular hostcell or, if from the same source, is modified from its original form.Thus, a heterologous gene in a host cell includes a gene that isendogenous to the particular host cell but has been modified through,for example, the use of DNA shuffling. The terms also includenon-naturally occurring multiple copies of a naturally occurring DNAsequence. Thus, the terms refer to a DNA segment that is foreign orheterologous to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is not ordinarilyfound. Exogenous DNA segments are expressed to yield exogenouspolypeptides.

“Inducible promoter” refers to those regulated promoters that can beturned on in a cell by an external stimulus, such as a chemical, light,hormone, stress, or a pathogen.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.,further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

The term “intracellular localization sequence” refers to a nucleotidesequence that encodes an intracellular targeting signal. An“intracellular targeting signal” is an amino acid sequence that istranslated in conjunction with a protein and directs it to a particularsub-cellular compartment. “Endoplasmic reticulum (ER) stop transitsignal” refers to a carboxy-terminal extension of a polypeptide, whichis translated in conjunction with the polypeptide and causes a proteinthat enters the secretory pathway to be retained in the ER. “ER stoptransit sequence” refers to a nucleotide sequence that encodes the ERtargeting signal.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. In the context of the present invention,an “isolated” or “purified” polynucleic acid (polynucleotide) segment oran “isolated” or “purified” polypeptide is a polynucleic acid segment orpolypeptide that, by the hand of man, exists apart from its nativeenvironment and is therefore not a product of nature. An isolatedpolynucleic acid segment or polypeptide may exist in a purified form ormay exist in a non-native environment such as, for example, a transgenichost cell. For example, an “isolated” or “purified” polynucleic acidsegment or protein, or biologically active portion thereof, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Preferably,an “isolated” polynucleic acid is free of sequences (preferably proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid molecule cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kbof nucleotide sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived. Aprotein that is substantially free of cellular material includespreparations of protein or polypeptide having less than about 30%, 20%,10%, 5%, (by dry weight) of contaminating protein. When the protein ofthe invention, or biologically active fragment (e.g., catalytically)thereof, is recombinantly produced, preferably culture medium representsless than about 30%, 20%, 10%, or 5% (by dry weight) of chemicalprecursors or non-protein-of-interest chemicals. Fragments and variantsof the disclosed nucleotide sequences and proteins or partial-lengthproteins encoded thereby are also encompassed by the present invention.By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence, and hence a portion of thepolypeptide or protein, encoded thereby.

A “marker gene” encodes a selectable or screenable trait.

The term “mature” protein refers to a post-translationally processedpolypeptide without its signal peptide. “Precursor” protein refers tothe primary product of translation of an mRNA. “Signal peptide” refersto the amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into the secretory pathway. The term“signal sequence” refers to a nucleotide sequence that encodes thesignal peptide.

The term “native gene” refers to gene that is present in the genome ofan untransformed cell.

“Naturally occurring” is used to describe an object that can be found innature as distinct from being artificially produced by man. For example,a protein or nucleotide sequence present in an organism (including avirus), which can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory, is naturallyoccurring.

The term “polynucleotide”, “nucleic acid”, “polynucleic acid” or“polynucleic acid segment” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., 1991; Ohtsuka et al., 1985;Rossolini et al., 1994).

NOV9X and Nov9X are used interchangeably herein.

A “nucleic acid fragment” is a fraction of a given nucleic acidmolecule. Deoxyribonucleic acid (DNA) is the genetic material whileribonucleic acid (RNA) is involved in the transfer of informationcontained within DNA into proteins. A “genome” is the entire body ofgenetic material contained in each cell of an organism. The term“nucleotide sequence” refers to a polymer of DNA or RNA which can besingle- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases capable of incorporation into DNA or RNApolymers. The terms “nucleic acid” or “nucleic acid sequence” may alsobe used interchangeably with gene, cDNA, DNA and RNA encoded by a gene(Batzer et al., 1991; Ohtsuka et al., 1985; Rossolini et al., 1999).Expression cassettes employed to introduce a phytase encoding openreading frame of the invention to a host cell preferably comprise atranscriptional initiation region linked to the open reading frame. Suchan expression cassette may be provided with a plurality of restrictionsites for insertion of the open reading frame and/or other DNAs, e.g., atranscriptional regulatory regions and/or selectable marker gene(s).

The transcriptional cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region,the DNA sequence of interest, and a transcriptional and translationaltermination region functional in a microbial cell. The terminationregion may be native with the transcriptional initiation region, may benative with the DNA sequence of interest, or may be derived from anothersource.

The terms “open reading frame” and “ORF” refer to the amino acidsequence encoded between translation initiation and termination codonsof a coding sequence. The terms “initiation codon” and “terminationcodon” refer to a unit of three adjacent nucleotides (‘codon’) in acoding sequence that specifies initiation and chain termination,respectively, of protein synthesis (mRNA translation).

“Operably linked” when used with respect to nucleic acid, means joinedas part of the same nucleic acid molecule, suitably positioned andoriented for transcription to be initiated from the promoter. DNAoperably linked to a promoter is “under transcriptional initiationregulation” of the promoter. Coding sequences can be operably-linked toregulatory sequences in sense or antisense orientation. When used withrespect to polypeptides, “operably linked” means joined as part of thesame polypeptide, i.e., via peptidyl bonds.

“Overexpression” refers to the level of expression in transgenic cellsor organisms that exceeds levels of expression in normal oruntransformed cells or organisms.

Known methods of polymerase chain reaction “PCR” include, but are notlimited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially mismatched primers, and the like. Seealso Innis et al., 1995; and Gelfand, 1995; and Innis and Gelfand, 1999.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA-box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence or functional RNA. This type of promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue specificity of a promoter. It is capable ofoperating in both orientations (normal or flipped), and is capable offunctioning even when moved either upstream or downstream from thepromoter. Both enhancers and other upstream promoter elements bindsequence-specific DNA-binding proteins that mediate their effects.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even be comprised of synthetic DNA segments. A promoter mayalso contain DNA sequences that are involved in the binding of proteinfactors which control the effectiveness of transcription initiation inresponse to physiological or developmental conditions.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor or factors, the minimalpromoter functions to permit transcription. A “minimal or core promoter”thus consists only of all basal elements needed for transcriptioninitiation, e.g., a TATA box and/or an initiator.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally- and/or spatially-regulated manner,and include both tissue-specific and inducible promoters. It includesnatural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. Different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions.

“Regulatory sequences” and “suitable regulatory sequences” each refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences includeenhancers, promoters, translation leader sequences, introns, andpolyadenylation signal sequences. They include natural and syntheticsequences as well as sequences which may be a combination of syntheticand natural sequences. As is noted above, the term “suitable regulatorysequences” is not limited to promoters. Some suitable regulatorysequences useful in the present invention will include, but are notlimited to, constitutive promoters inducible promoters and viralpromoters.

The term “RNA transcript” refers to the product resulting from RNApolymerase catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” (mRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a single- or a double-stranded DNA that iscomplementary to and derived from mRNA.

“Stably transformed” refers to cells that have been selected andregenerated on a selection media following transformation.

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

“Transcription stop fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as polyadenylation signalsequences, capable of terminating transcription.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell. Host cells containing thetransformed nucleic acid fragments are referred to as “transgenic”cells.

“Transformed,” “transgenic,” and “recombinant” refer to a host cell suchas a bacterium into which a heterologous nucleic acid molecule has beenintroduced. The nucleic acid molecule can be stably integrated into thegenome by methods generally known in the art which are disclosed inSambrook et al., 1989). For example, “transformed,” “transformant,” and“transgenic” cells have been through the transformation process andcontain a foreign gene, e.g., as an episomal element or integrated intotheir chromosome. The term “untransformed” refers to cells that have notbeen through the transformation process.

A “transgene” refers to a gene that has been introduced into the genomeby transformation and is stably maintained. Transgenes may include, forexample, genes that are either heterologous or homologous to the genesof a particular cell to be transformed. Additionally, transgenes maycomprise native genes inserted into a non-native organism, or chimericgenes. The term “endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host cell but that is introduced by genetransfer.

“Transiently transformed” refers to cells in which an expressioncassette, polynucleotide or transgene has been introduced but notselected for stable maintenance.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

“Translation stop fragment” refers to nucleotide sequences that containone or more regulatory signals, such as one or more termination codonsin all three frames, capable of terminating translation. Insertion of atranslation stop fragment adjacent to or near the initiation codon atthe 5′ end of the coding sequence will result in no translation orimproper translation. Excision of the translation stop fragment bysite-specific recombination will leave a site-specific sequence in thecoding sequence that does not interfere with proper translation usingthe initiation codon.

A polypeptide or enzyme exhibiting “phytase” activity or a “phytase” isintended to cover any enzyme capable of effecting the liberation ofinorganic phosphate or phosphorous from various myo-inositol phosphates.Examples of such myo-inositol phosphates (phytase substrates) are phyticacid and any salt thereof, e.g., sodium phytate or potassium phytate ormixed salts. Also any stereoisomer of the mono-, di-, tri-, tetra- orpenta-phosphates of myo-inositol may serve as a phytase substrate. Inaccordance with the above definition, the phytase activity can bedetermined using any assay in which one of these substrates is used. Athermotolerant phytase of the invention includes variant polypeptidesderived from a particular thermotolerant phytase by deletion (so-calledtruncation) or addition of one or more amino acids to the N-terminaland/or C-terminal end of the native protein; deletion or addition of oneor more amino acids at one or more sites in the native protein; orsubstitution of one or more amino acids at one or more sites in thethermotolerant phytase. Such variants may result from, for example, fromhuman manipulation. Methods for such manipulations are generally knownin the art. For example, amino acid sequence variants of thepolypeptides can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel, 1985; Kunkel et al., 1987; U.S. Pat. No.4,873,192; Walker and Gaastra, 1983, and the references cited therein.Guidance as to appropriate amino acid substitutions that do not affectbiological activity of the protein of interest may be found in the modelof Dayhoff et al., 1978, herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, are preferred.

Thus, the thermotolerant phytase genes and nucleotide sequences of theinvention include both the naturally occurring sequences as well asmutant forms. Likewise, the thermotolerant phytase polypeptides of theinvention encompass both naturally occurring proteins as well asvariations and modified forms thereof. Such variants will continue topossess the desired activity. The deletions, insertions, andsubstitutions of the polypeptide sequence encompassed herein are notexpected to produce radical changes in the characteristics of thepolypeptide. Nevertheless, one skilled in the art will appreciate thatthe effect will be evaluated by routine screening assays. The nucleicacid molecules of the invention can be optimized for enhanced expressionin a host cell of interest. It is recognized that all or any part of thegene sequence may be optimized or synthetic. That is, synthetic orpartially optimized sequences may also be used. Variant nucleotidesequences and proteins also encompass sequences and protein derived froma mutagenic and recombinogenic procedure such as DNA shuffling. Withsuch a procedure, one or more different coding sequences can bemanipulated to create a new polypeptide possessing the desiredproperties. In this manner, libraries of recombinant polynucleotides aregenerated from a population of related sequence polynucleotidescomprising sequence regions that have substantial sequence identity andcan be homologously recombined in vitro or in vivo. Strategies for suchDNA shuffling are known in the art. See, for example, Stemmer, 1994;Stemmer, 1994; Crameri et al., 1997; Moore et al., 1997; Zhang et al.,1997; Crameri et al., 1998; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

By “variants” is intended substantially similar sequences. Fornucleotide sequences, variants include those sequences that, because ofthe degeneracy of the genetic code, encode the identical amino acidsequence of the reference protein. Naturally occurring allelic variantssuch as these can be identified with the use of well-known molecularbiology techniques, as, for example, with polymerase chain reaction(PCR) and hybridization techniques. Variant nucleotide sequences alsoinclude synthetically derived nucleotide sequences, such as thosegenerated, for example, by using site-directed mutagenesis which encodethe reference protein, as well as those that encode a polypeptide havingamino acid substitutions. Generally, nucleotide sequence variants of theinvention will have at least 40%, 50%, 60%, preferably 70%, morepreferably 80%, even more preferably 90%, most preferably 99%, andsingle unit percentage identity to the native nucleotide sequence basedon these classes. For example, 71%, 72%, 73% and the like, up to atleast the 90% class. Variants may also include a full length genecorresponding to an identified gene fragment.

“Vector” is defined to include, inter alia, any plasmid, cosmid, phageor other vector in double or single stranded linear or circular formwhich may or may not be self transmissible or mobilizable, and which cantransform prokaryotic or eukaryotic host either by integration into thecellular genome or exist extrachromosomally (e.g., autonomousreplicating plasmid with an origin of replication).

Preferred Constructs and Host Cells of the Invention

The invention preferably provides an expression cassette which comprisesa nucleic acid sequence (promoter) capable of directing expression of apolynucleotide encoding a thermotolerant phytase either in vitro or invivo. Methods to prepare and/or identify a thermotolerant phytaseinclude mutagenesis, e.g., recursive mutagenesis, and/or selection orscreening, e.g., for phytases having activity at temperatures greaterthan 60° C. Methods for mutagenesis and nucleotide sequence alterationsare well known in the art. See, for example, Kunkel, 1985; Kunkel etal., 1987; U.S. Pat. No. 4,873,192; Walker and Gaastra, 1983 and thereferences cited therein; and Arnold et al., 1996.

A. DNA and Host Cells for Transformation

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) BACs(bacterial artificial chromosomes) and DNA segments for use intransforming cells will generally comprise the phytase encoding DNA, aswell as other DNA such as cDNA, gene or genes which one desires tointroduce into the cells. These DNA constructs can further includestructures such as promoters, enhancers, polylinkers, or even regulatorygenes as desired. One of the DNA segments or genes chosen for cellularintroduction will often encode a protein which will be expressed in theresultant transformed (recombinant) cells, such as will result in ascreenable or selectable trait and/or which will impart an improvedphenotype to the transformed cell. However, this may not always be thecase, and the present invention also encompasses transformed cellsincorporating non-expressed transgenes.

DNA useful for introduction into cells includes that which has beenderived or isolated from any source, that may be subsequentlycharacterized as to structure, size and/or function, chemically altered,and later introduced into cells. An example of DNA “derived” from asource, would be a DNA sequence that is identified as a useful fragmentwithin a given organism, and which is then chemically synthesized inessentially pure form. An example of such DNA “isolated” from a sourcewould be a useful DNA sequence that is excised or removed from saidsource by chemical means, e.g., by the use of restriction endonucleases,so that it can be further manipulated, e.g., amplified, for use in theinvention, by the methodology of genetic engineering. Such DNA iscommonly referred to as “recombinant DNA.”

Therefore useful DNA includes completely synthetic DNA, semi-syntheticDNA, DNA isolated from biological sources, and DNA derived fromintroduced RNA. Generally, the introduced DNA is not originally residentin the genotype which is the recipient of the DNA, but it is within thescope of the invention to isolate a gene from a given genotype, and tosubsequently introduce multiple copies of the gene into the samegenotype, e.g., to enhance production of a given gene product.

The introduced DNA includes, but is not limited to, DNA from genes suchas those from bacteria, yeasts, fungi, or viruses. The introduced DNAcan include modified or synthetic genes, portions of genes, or chimericgenes, including genes from the same or different genotype. The term“chimeric gene” or “chimeric DNA” is defined as a gene or DNA sequenceor segment comprising at least two DNA sequences or segments fromspecies which do not combine DNA under natural conditions, or which DNAsequences or segments are positioned or linked in a manner which doesnot normally occur in the native genome of the untransformed cell.

The introduced DNA used for transformation herein may be circular orlinear, double-stranded or single-stranded. Generally, the DNA is in theform of chimeric DNA, such as plasmid DNA, that can also contain codingregions flanked by regulatory sequences which promote the expression ofthe recombinant DNA present in the transformed cell. For example, theDNA may itself comprise or consist of a promoter that is active in acell which is derived from a source other than that cell, or may utilizea promoter already present in the cell that is the transformationtarget.

Generally, the introduced DNA will be relatively small, i.e., less thanabout 30 kb to minimize any susceptibility to physical, chemical, orenzymatic degradation which is known to increase as the size of the DNAincreases. The number of proteins, RNA transcripts or mixtures thereofwhich is introduced into the cell is preferably preselected and defined,e.g., from one to about 5-10 such products of the introduced DNA may beformed.

The selection of an appropriate expression vector will depend upon thehost cells. Typically an expression vector contains (1) prokaryotic DNAelements coding for a bacterial origin of replication and an antibioticresistance gene to provide for the amplification and selection of theexpression vector in a bacterial host; (2) DNA elements that controlinitiation of transcription such as a promoter; (3) DNA elements thatcontrol the processing of transcripts such as introns, transcriptiontermination/polyadenylation sequence; and (4) a gene of interest that isoperatively linked to the DNA elements to control transcriptioninitiation. The expression vector used may be one capable ofautonomously replicating in the above host or capable of integratinginto the chromosome, originally containing a promoter at a site enablingtranscription of the linked phytase gene.

If prokaryotes such as bacteria are used as the host, the expressionvector for the phytase is preferably one capable of autonomouslyreplicating in the micro-organism and comprising a promoter, aribosome-binding sequence, the novel phytase gene, and a transcriptiontermination sequence. The vector may also contain a gene for regulatingthe promoter.

Yeast or fungal expression vectors may comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences.

Suitable vectors include by way of example: for bacteria, pQE70, pQE60,pQE-9 (Qiagen), pBluescript II (Stratagene), pTRC99a, pKK223-3, pDR540,pRIT2T (Pharmacia); for eukaryotic cells: pXT1, pSG5 (Stratagene) pSVK3,pBPV, pMSG, pSVLSV40 (Pharmacia). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1(Promega Biotec, Madison, Wis., USA). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Bacillus subtilis; andvarious species within the genera Escherichia, Pseudomonas, Serratia,Streptomyces, Corynebacterium, Brevibacterium, Bacillus, Microbacterium,and Staphylococcus, although others may also be employed as a matter ofchoice; fungal cells belonging to the genera Aspergillus, Rhizopus,Trichoderma, Neurospora, Mucor, Penicillium, etc., such as yeastbelonging to the genera Kluyveromyces, Saccharomyces,Schizosaccharomyces, Trichosporon, Schwanniomyces, and the like.

The construction of vectors which may be employed in conjunction withthe present invention will be known to those of skill of the art inlight of the present disclosure (see, e.g., Sambrook et al., 1989;Gelvin et al., 1990). The expression cassette of the invention maycontain one or a plurality of restriction sites allowing for placementof the polynucleotide encoding a thermotolerant phytase under theregulation of a regulatory sequence. The expression cassette may alsocontain a termination signal operably linked to the polynucleotide aswell as regulatory sequences required for proper translation of thepolynucleotide. The expression cassette containing the polynucleotide ofthe invention may be chimeric, meaning that at least one of itscomponents is heterologous with respect to at least one of the othercomponents. Expression of the polynucleotide in the expression cassettemay be under the control of a constitutive promoter, inducible promoter,regulated promoter, viral promoter or synthetic promoter.

The expression cassette may include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region,the polynucleotide of the invention and a transcriptional andtranslational termination region functional in vivo and/or in vitro. Thetermination region may be native with the transcriptional initiationregion, may be native with the polynucleotide, or may be derived fromanother source. The regulatory sequences may be located upstream (5′non-coding sequences), within (intron), or downstream (3′ non-codingsequences) of a coding sequence, and influence the transcription, RNAprocessing or stability, and/or translation of the associated codingsequence. Regulatory sequences may include, but are not limited to,enhancers, promoters, repressor binding sites, translation leadersequences, introns, and polyadenylation signal sequences. They mayinclude natural and synthetic sequences as well as sequences which maybe a combination of synthetic and natural sequences.

The vector, used in the present invention may also include appropriatesequences for amplifying expression.

B. Regulatory Sequences

A promoter is a nucleotide sequence which controls the expression of acoding sequence by providing the recognition for RNA polymerase andother factors required for proper transcription. A promoter includes aminimal promoter, consisting only of all basal elements needed fortranscription initiation, such as a TATA-box and/or initiator that is ashort DNA sequence comprised of a TATA-box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. A promoter maybe derived entirely from a native gene, or be composed of differentelements derived from different promoters found in nature, or even becomprised of synthetic DNA segments. A promoter may contain DNAsequences that are involved in the binding of protein factors whichcontrol the effectiveness of transcription initiation in response tophysiological or developmental conditions. A promoter may also include aminimal promoter plus a regulatory element or elements capable ofcontrolling the expression of a coding sequence or functional RNA. Thistype of promoter sequence contains of proximal and more distal elements,the latter elements are often referred to as enhancers.

Representative examples of promoters include, but are not limited to,promoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses. Particular bacterial promotersinclude E. coli lac or trp, the phage lambda P_(L), lac, lacZ, T3, T7,gpt, and lambda P_(R) promoters.

Any promoter capable of expressing in yeast hosts can be used as thepromoter. Examples thereof include promoters for genes of hexokinase andthe like in the glycolytic pathway, and promoters such as gal 1promoter, gal 10 promoter, heat shock protein promoter, MFa-1 promoterand CUP 1 promoter.

Any promoter capable of expressing in filamentous fungi may be used.Examples are a promoter induced strongly by starch or cellulose, e.g., apromoter for glucoamylase or a-amylase from the genus Aspergillus orcellulase (cellobiohydrase) from the genus Trichoderma, a promoter forenzymes in the glycolytic pathway, such as phosphoglycerate kinase (pgk)and glycerylaldehyde 3-phosphate dehydrogenase (gpd), etc.

Two principal methods for the control of expression are known, viz.:overexpression and underexpression. Overexpression can be achieved byinsertion of one or more than one extra copy of the selected gene. Forunderexpression there are two principle methods which are commonlyreferred to in the art as “antisense downregulation” and “sensedownregulation”. Generically these processes are referred to as “genesilencing”. Both of these methods lead to an inhibition of expression ofthe target gene.

Several inducible promoters are known in the art. Many are described ina review by Gatz (1996) (see also Gatz, 1997). Examples includetetracycline repressor system, Lac repressor system, copper-induciblesystems, salicylate-inducible systems (such as the PR1a system),glucocorticoid-inducible (Aoyama T. et al., 1997) and ecdysome-induciblesystems. Also included are the benzene sulphonamide-inducible (U.S. Pat.No. 5,364,780) and alcohol-inducible (WO 97/06269 and WO 97/06268)inducible systems and glutathione S-transferase promoters.

Regulated expression of a chimeric transacting viral replication proteincan be further regulated by other genetic strategies. For example,Cre-mediated gene activation as described by Odell et al., 1990. Thus, aDNA fragment containing 3′ regulatory sequence bound by lox sitesbetween the promoter and the replication protein coding sequence thatblocks the expression of a chimeric replication gene from the promotercan be removed by Cre-mediated excision and result in the expression ofthe trans-acting replication gene. In this case, the chimeric Cre gene,the chimeric trans-acting replication gene, or both can be under thecontrol of developmental-specific or inducible promoters. An alternategenetic strategy is the use of tRNA suppressor gene. For example, theregulated expression of a tRNA suppressor gene can conditionally controlexpression of a trans-acting replication protein coding sequencecontaining an appropriate termination codon as described by Ulmasov etal., 1997. Again, either the chimeric tRNA suppressor gene, the chimerictransacting replication gene, or both can be under the control ofdevelopmental-specific or inducible promoters.

In addition to the use of a particular promoter, other types of elementscan influence expression of transgenes. In particular, introns havedemonstrated the potential for enhancing transgene expression.

Other elements include those that can be regulated by endogenous orexogenous agents, e.g., by zinc finger proteins, including naturallyoccurring zinc finger proteins or chimeric zinc finger proteins. See,e.g., U.S. Pat. No. 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO98/53057; WO 98/53058; WO 00/23464; WO 95/19431; and WO 98/54311.

An enhancer is a DNA sequence which can stimulate promoter activity andmay be an innate element of the promoter or a heterologous elementinserted to enhance the level or tissue specificity of a particularpromoter. An enhancer is capable of operating in both orientations (5′to 3′ and 3′-5′ relative to the gene of interest coding sequences), andis capable of functioning even when moved either upstream or downstreamfrom the promoter. Both enhancers and other upstream promoter elementsbind sequence-specific DNA-binding proteins that mediate their effects.

Vectors for use in accordance with the present invention may beconstructed to include an enhancer element. Constructs of the inventionwill also include the gene of interest along with a 3′ end DNA sequencethat acts as a signal to terminate transcription and allow for thepolyadenylation of the resultant mRNA.

As the DNA sequence between the transcription initiation site and thestart of the coding sequence, i.e., the untranslated leader sequence,can influence gene expression, one may also wish to employ a particularleader sequence. Preferred leader sequences are contemplated to includethose which include sequences predicted to direct optimum expression ofthe attached gene, i.e., to include a preferred consensus leadersequence which may increase or maintain mRNA stability and preventinappropriate initiation of translation. The choice of such sequenceswill be known to those of skill in the art in light of the presentdisclosure.

C. Marker Genes

In order to improve the ability to identify transformants, one maydesire to employ a selectable or screenable marker gene as, or inaddition to, the expressible gene of interest. “Marker genes” are genesthat impart a distinct phenotype to cells expressing the marker gene andthus allow such transformed cells to be distinguished from cells that donot have the marker. Such genes may encode either a selectable orscreenable marker, depending on whether the marker confers a trait whichone can ‘select’ for by chemical means, i.e., through the use of aselective agent (e.g., an antibiotic, or the like), or whether it issimply a trait that one can identify through observation or testing,i.e., by ‘screening’. Of course, many examples of suitable marker genesare known to the art and can be employed in the practice of theinvention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes which can bedetected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA and small active enzymes detectable in extracellularsolution.

Selectable markers for use in prokaryotes include a tetracyclineresistance or an ampillicin resistance gene. Screenable markers that maybe employed include, but are not limited to, a b-glucuronidase or uidAgene (GUS) which encodes an enzyme for which various chromogenicsubstrates are known; a beta-lactamase gene (Sutcliffe, 1978), whichencodes an enzyme for which various chromogenic substrates are known(e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky etal., 1983) which encodes a catechol dioxygenase that can convertchromogenic catechols; an alpha-amylase gene (Ikuta et al., 1990); atyrosinase gene (Katz et al., 1983) which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone which in turn condenses toform the easily detectable compound melanin; a beta-galactosidase gene,which encodes an enzyme for which there are chromogenic substrates; aluciferase (lux) gene (Ow et al., 1986), which allows forbioluminescence detection; or even an aequorin gene (Prasher et al.,1985), which may be employed in calcium-sensitive bioluminescencedetection, or a green fluorescent protein gene (Niedz et al., 1995).

Transformation

The expression cassette, or a vector construct containing the expressioncassette, may be inserted into a cell. The expression cassette or vectorconstruct may be carried episomally or integrated into the genome of thecell, e.g., derivatives of SV40; bacterial plasmids; phage DNA;baculovirus; yeast plasmids; vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. However, any vector may be used as long as itis replicable and viable in the host.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a cellular host. Transformationof microbial cells may be accomplished through use of polyethyleneglycol, calcium chloride, viral infection, DEAE dextran, phageinfection, electroporation and other methods known in the art.Transformation of fungus may be accomplished according to Gonni et al.(1987). Introduction of the recombinant vector into yeasts can beaccomplished by methods including electroporation, use of spheroplasts,lithium acetate, and the like. Any method capable of introducing DNAinto animal cells can be used: for example, electroporation, calciumphosphate, lipofection and the like.

Recombinant Enzyme

For preparation of recombinant phytase, following transformation of asuitable host strain and growth of the host strain to an appropriatecell density, e.g., a bacterial or yeast host, a selected promoter maybe induced by appropriate means (e.g., temperature shift or chemicalinduction) and cells cultured for an additional period to yieldrecombinant enzyme. Cells are then typically harvested bycentrifugation, disrupted by physical or chemical means, and theresulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

The enzyme can be recovered and purified from recombinant cell culturesby methods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The enzymes of the present invention may be a product of chemicalsynthetic procedures, or produced by recombinant techniques from amicrobial host (for example, by bacterial, yeast, and fungal cells inculture). Depending upon the host employed in a recombinant productionprocedure, the enzyme of the present invention may or may not becovalently modified via glycosylation. In eukaryotic cells,glycosylation of secreted proteins serves to modulate protein folding,conformational and thermostability stability, and resistance toproteolysis. Given a specific application of phytase use, a glycosylatedversion of the enzyme may be preferable over a non-glycosylated form.For example, the use of a glycosylated phytase in animal feed helpsprotect the enzyme from thermal denaturation during feed pelleting andfrom proteolytic inactivation as it passes through the stomach of theanimal, helping deliver active enzyme to the intestinal tract and siteof action. For food processing applications where enzyme activity isdesired only during processing and not in the final product anon-glycosylated, thermolabile, and proteolytic susceptible phytase ispreferred. By producing the phytase of this invention in variousmicrobial hosts, both thermotolerance and susceptibility to proteolyticdegradation are altered. For example, when produced in Escherichia colithe phytase of the present invention exhibits a half life of 8.4 minutesin simulated gastric fluid, while in Pichia pastoris andSchizosaccharomyces pombe these values increase to 10.4 and 29.2minutes, respectively. E. coli does not posses the cellular machinery toglycosylate proteins, while the extent of glycosylation in S. pombe isgreater than in P. pastoris. Similarly, residual activity following a 5minute heating step at 95° C. increases with increasing degrees ofglycosylation. In E. coli 10% residual activity is measured, while in P.pastoris and S. pombe the values increase to 30 and 50%, respectively.Enzymes of the invention may or may not also include an initialmethionine amino acid residue.

The enzymes of this invention may be employed for any purpose in whichsuch enzyme activity is necessary or desired. In a preferred embodiment,the enzyme is employed for catalyzing the hydrolysis of phytate inanimal feed. In another preferred embodiment, the enzyme is employed forcatalyzing the hydrolysis of phytate in food.

Phytase Compositions

Generally, phytase compositions are liquid or dry. Liquid compositionsneed not contain anything more than the phytase enzyme, preferably in ahighly purified form. However, a stabilizer such as glycerol, sorbitolor mono propylen glycol may be added. The liquid composition may alsocomprise other additives, such as salts, sugars, preservatives,pH-adjusting agents, proteins, and phytate (a phytase substrate).Typical liquid compositions are aqueous or oil-based slurries. Theliquid compositions may be added to a food or feed before or after anoptional pelleting thereof.

Dry compositions may be freeze-dried or spray dried compositions, inwhich case the composition need not contain anything more than theenzyme in a dry form. Dry compositions may be granulates which mayreadily be mixed with, e.g., food or feed components, or morepreferably, form a component of a pre-mix. The particle size of theenzyme granulates preferably is compatible with that of the othercomponents of the mixture. This provides a safe and convenient means ofincorporating enzymes into, e.g., processed food or animal feed.

For example, a stable phytase enzyme formulation can be prepared byfreezing a mixture of liquid enzyme solution with a bulking agent suchas ground soybean meal, and then lyophilizing the mixture. The reductionin moisture and the binding interactions of the phytase with the bulkingagent protect the enzyme from external environmental factors such as thetemperature extremes experienced during compound feed manufacture. Dryformulations can further enhance stability by minimizing the activity ofpotential proteolytic enzymes that may be present as by-products in theliquid fermentation mixture used to manufacture the target enzyme. Theresulting dry enzyme-soy flour mixture of the present invention canwithstand high extremes of temperature. For example, after 120 minutesof heating at 96° C., the dry enzyme formulation retained 97.8% of itsoriginal enzymatic activity. This formulated enzyme mixture can be usedas a feed supplement for use in poultry and swine production. Forinstance, addition of 500 enzyme units of a thermotolerant phytase ofthe invention to 1 kg of a standard corn-soy poultry diet allowed areduction in the levels of inorganic phosphate supplementation currentlyused in animal nutrition, i.e., from 0.45% to 0.225%. Chickens raised ona 0.225% phosphate diet supplemented with the formulated phytaseperformed as well as birds fed a standard diet containing 0.45%phosphate. Moreover, a reduction in phosphate supplementation results indecreased levels of phosphate pollution, which in turn significantlylessens the environmental impact of intensive commercial animalproduction.

Once a dry enzyme preparation is obtained, agglomeration granulates areprepared using agglomeration techniques in a high shear mixer duringwhich a filler material and the enzyme are co-agglomerated to formgranules. Absorption granulates are prepared by having cores of acarrier material to absorb/be coated by the enzyme. Typical fillermaterials are salts such as disodium sulphate. Other fillers includekaolin, talc, magnesium aluminium silicate and cellulose fibres.Optionally, binders such as dextrins are also included in agglomerationgranulates.

Typical carrier materials include starch, e.g., in the form of cassava,corn, potato, rice and wheat. Salts may also be used.

Optionally, the granulates are coated with a coating mixture. Such amixture comprises coating agents, preferably hydrophobic coating agents,such as hydrogenated palm oil and beef tallow, and if desired, otheradditives such as calcium carbonate or kaolin.

Additionally, phytase compositions may contain other substituents suchas coloring agents, aroma compounds, stabilizers, vitamins, minerals,other feed or food enhancing enzymes and the like. This is so inparticular for the so-called pre-mixes.

A “food or feed additive” is an essentially pure compound or a multicomponent composition intended for or suitable for being added to foodor feed. In particular it is a substance that by its intended use isbecoming a component of a food or feed product or affects anycharacteristics of a food or feed product. Thus, a phytase additive isunderstood to mean a phytase which is not a natural constituent of themain feed or food substances or is not present at its naturalconcentration therein, e.g., the phytase is added to the feed separatelyfrom the feed substances, alone or in combination with other feedadditives. A typical additive usually comprises one or more compoundssuch as vitamins, minerals or feed enhancing enzymes and suitablecarriers and/or excipients.

A “ready for use” phytase additive is herein defined as an additive thatis not produced in situ in animal feed or in processed food. A ready foruse phytase additive may be fed to humans or animals directly or,preferably, directly after mixing with other feed or food constituents.For example, a feed additive according to this aspect of the presentinvention is combined with other feed components to produce feed. Suchother feed components include one or more other (preferablythermostable) enzyme supplements, vitamin feed additives, mineral feedadditives and amino acid feed additives. The resulting (combined) feedadditive including possibly several different types of compounds canthen be mixed in an appropriate amount with the other feed componentssuch as cereal and protein supplements to form an animal feed.Processing of these components into an animal feed can be performedusing any of the currently used processing apparatuses such as adouble-pelleting machine, a steam pelleter, an expander or an extruder.

Similarly, a food additive according to this aspect of the presentinvention is combined with other food components to produce processedfood products. Such other food components include one or more other(preferably thermostable) enzyme supplements, vitamin food additives andmineral food additives. The resulting (combined) food additive,including possibly several different types of compounds can then bemixed in an appropriate amount with the other food components such ascereal and plant proteins to form a processed food product. Processingof these components into a processed food product can be performed usingany of the currently used processing apparatuses.

In a preferred embodiment, the phytase compositions of the inventionadditionally comprises an effective amount of one or more feed or foodenhancing enzymes, in particular feed or food enhancing enzymes selectedfrom the group consisting of alpha-galactosidases, beta-galactosidases,in particular lactases, other phytases, beta-glucanases, in particularendo-beta-1,4-glucanases and endo-beta-1,3(4)-glucanases, cellulases,xylosidases, galactanases, in particular arabinogalactanendo-1,4-beta-galactosidases and arabinogalactanendo-1,3-beta-galactosidases, endoglucanases, in particularendo-1,2-beta-glucanase, endo-1,3-alpha-glucanase, andendo-1,3-beta-glucanase, pectin degrading enzymes, in particularpectinases, pectinesterases, pectin lyases, polygalacturonases,arabinanases, rhamnogalacturonases, rhamnogalacturonan acetyl esterases,rhamnogalacturonan-alpha-rhamnosidase, pectate lyases, andalpha-galacturonisidases, mannanases, beta-mannosidases, mannan acetylesterases, xylan acetyl esterases, proteases, xylanases,arabinoxylanases and lipolytic enzymes such as lipases, phospholipasesand cutinases.

The animal feed additive of the invention is supplemented to the animalbefore or simultaneously with the diet. Preferably, the animal feedadditive of the invention is supplemented to the animal simultaneouslywith the diet.

An effective amount of phytase in food or feed is from about 10 to20,000 FTU/kg; preferably from about 10 to 15,000 FTU/kg, morepreferably from about 10 to 10,000 FTU/kg, in particular from about 100to 5,000 FTU/kg, especially from about 100 to about 2,000 FTU/kg feed orfood.

Also within the scope of this invention is the use of phytase forprocessing and manufacturing human foods and animal feeds. Grains andflours destined for human foods can be enzymatically treated withphytase to reduce the phytin content of the material. The reduced levelsof phytin enhance the quality of the food by increasing the nutrientavailability of essential minerals such as iron, calcium, and zinc. Inaddition to increasing the nutritional quality of food, phytase usedduring food processing can improve the overall efficiency of the foodproduction method. For example, addition of phytase to white soybeanflakes during soy protein isolate manufacturing can significantlyincrease the yield and quality of extractable protein. During foodmanufacture the phytase is active during manufacture and processingonly, and is not active in the final food product. This aspect isrelevant for instance in dough making and baking. Similarly, animal feedgrains such as toasted soybean meal or canola meal may be pre-processedwith phytase prior to compound feed manufacture. Removal of theanti-nutritive factors in animal feed components prior to compound feedmanufacture produces a nutritionally higher quality and more valuableanimal feed ingredient. In this processing method the phytase is activeduring feed manufacturing, and may or may not be active in the digestivetract of the animal upon ingestion of the treated feed.

In addition to using phytase as a food processing aid, the scope of thisinvention encompasses the use of phytase as a human supplementaldigestive aid. Phytase in tablet form can be ingested at the time offood consumption to deliver active enzyme to the gastrointestinal tractof the recipient. Nutritional gains for the consumer would beexperienced in vivo and may be taken with foods that cannot be treatedwith a phytase during food processing.

Also within the scope of the invention is the use of a phytase of theinvention during the preparation of food or feed preparations oradditives, i.e., the phytase is active during the manufacture only andis not active in the final food or feed product. This aspect isparticularly relevant, for instance, in dough making and baking and theproduction of other ready-to-eat cereal based products.

The phytase may also be used advantageously in monogastrics as well asin polygastrics, especially young calves. Diets for fish and crustaceansmay also be supplemented with phytase to further improve feed conversionratio and reduce the level of excreted phosphorus for intensiveproduction systems. The feed according to the present invention may alsobe provided to animals such as poultry, e.g., turkeys, geese, ducks, aswell as swine, equine, bovine, ovine, caprine, canine and feline, aswell as fish and crustaceans. It is however, particularly preferred thatthe feed is provided to pigs or to poultry, including, but not limitedto, broiler chickens, hens, in particular laying hens, turkeys andducks.

Feed Compositions and Methods of Use

The phytases (formulated as described above) of the current inventionmay be combined with other ingredients to result in novel feedcompositions with particular advantages.

For instance, it is preferable that intensive animal productionoperations limit the phosphate pollution that is contained in the fecesof the animals that are produced. The amount of phosphate present in thediet and the availability of the phosphate in the diet to the animal arethe primary factors influencing the excreted phosphate present in thefeces of the animal. Currently, the availability of the plant, orgrain-derived phosphate, present in soybean meal, corn grain (and otherfeedstuffs) is low as the phosphate is primarily in the form of phyticacid. In order to maximize the growth efficiencies of the animalsinorganic phosphate is added to feed resulting in a feed compositionthat contains adequate levels of available phosphate. However, thesefeed formulations contain too much total phosphate and result inphosphate pollution.

Although commercially available phytases at present result in higherphosphate availability they are recommended to be used with high levelsof added inorganic phosphate. The phytases of the present invention areso active that they can be used to create novel animal feed formulationsthat have a) significantly reduced levels of inorganic phosphate, and b)allow superior feed conversion efficiency and improved weight gainrelative to normal diets. At present, commercially available phytaseswill not allow animals to be efficiently produced on a feed thatcontains no added inorganic phosphorus

Specifically, the animal feed of the invention comprises the combinationof a phytase of the present invention in combination with animal feedingredients to form a feed that has substantially lowered inorganicphosphorus levels. In a preferred embodiment, the feed compositions ofthe invention comprises typical feed ingredients, micronutrients,vitamins, etc. and an effective amount of thermostable phytase andinorganic phosphate where the amounts of the phytase and phosphorus arefrom about between the levels of 50-20,000 units of phytase per kg offeed and less than 0.45% inorganic phosphorus; preferably between thelevels of 100-10,000 units of phytase per kg of feed and less than0.225% inorganic phosphorus; in particular between the levels of150-10,000 units of phytase per kg of feed and less than 0.15% inorganicphosphorus, or especially between the levels of 250-20,000 units ofphytase per kg of feed and no exogenously added inorganic phosphorus.

Also, within the scope of the invention are methods of improving weightgains, and feed conversions ratios (FCR) associated with production offarm animals. A phytase of the present invention allows improved weightgains and FCR especially when used in combination with diets that arelow in inorganic phosphate. Specifically the method of the presentinvention to improve the FCR, or weight gain of a low inorganicphosphate diet by feeding a diet to an animal comprising a phytase ofthe present invention and a level of inorganic phosphate at or below thelevel of 0.45%. Preferably, the method comprises feeding a dietcontaining the phytase and less than 0.225% inorganic phosphate, or mostpreferably the method comprises feeding a diet containing the phytaseand no added inorganic phosphorus.

The animal feed of the present invention can be used on monogastric orpolygastric animals. The animal feed of the present invention can befeed for poultry, or swine, or calves, or companion animals such as dogsor cats or horsed. Examples of such feed and the use of the feed areprovided in Example 3.

The present invention also provides for a method of animal husbandrythat results in a significantly reduced environmental phosphate load.The method comprises feeding entire flocks or herds of farm animals afeed composition containing a phytase of the present invention and areduced amount of inorganic phosphorus (less than 0.45%). Morepreferably the method comprises feeding entire flocks or herds of farmanimals a feed composition containing a phytase of the present inventionand a significantly reduced amount of inorganic phosphorus (less than0.225%), or most preferably the method comprising feeding entire flocksor herds of farm animals a feed composition containing a phytase of thepresent invention and no inorganic phosphorus. This method will allowhigh densities of animals to be maintained while minimizing theenvironmental release of phosphate from the farming operation.

The invention will be further described by the following examples, whichare not intended to limit the scope of the invention in any manner.

EXAMPLE 1 Exemplary Methods to Prepare and Identify ThermotolerantPhytases Recombinant Expression

For expression in Aspergillus niger, A. niger NW205 (ura-arg-nic-) maybe transformed and screened for phytase-producing transformants asdescribed in Passamontes et al. (1997).

For expression in Saccharomyces cerevisiae, a phytase gene is clonedinto a 2μ-based vector, such as one harboring a shortened version of thegap(FL) promoter and the pho5 terminator (Janes et al., 1990) as well asa selection marker. The phytase gene is cloned downstream of the gap(FL)promoter in the EcoRI-BamHI blunt-ended expression cassette. S.cerevisiae YMR4 (urg⁻ his⁻ leu⁻ pho3⁻ pho5⁻) is used for transformation.Individual transformants are grown initially for 1 to 2 days in minimalmedium. Phytase production is tested after subsequent culture for 2 to 3days in YPD medium.

For expression in Hansenula polymorpha, the phytase gene may be clonedas an EcoRI fragment into the corresponding site of the H. polymorphaexpression vector pFP (Gellisen et al., 1991) downstream of the formatedehydrogenase (FMD) promoter (EPA 299108). The resulting plasmid istransformed into H. polymorpha RB11. Transformants are individuallyinoculated into minimal medium (YNB containing 2% glucose). Afterseveral passages under selective pressure to force multiple integrationsof the expression plasmids into the genome of H. polymorpha, singlestable clones are tested for phytase activity.

For expression in Pichia, a pPIcαA vector encoding a phytase istransformed into P. pastoris strain X33 by electroporation according tothe manufacturer's instructions (Invitrogen). The transformed cells areplated into YPD-Zeocin agar medium and positive colonies are incubatedin minimal medium with glycerol (BMGY) for 24 hours. When the yeast celldensity reaches 2.5×10⁸ cells/ml (OD₆₀₀=5), the cells are centrifugedand suspended in 0.5% methanol medium (BMMY) to induce gene expression.

Protein Purification

Culture broths (typically 500 to 1,000 ml) are centrifuged to removecells and concentrated by ultrafiltration with Amicon 8400 cells (PM30membranes; Grace Ag, Wallisellen, Switzerland) and ultrafree-15centrifugal filter devices (Biomax-30K; Millipore, Bedford, Mass.). Theconcentrates (typically 1.5 to 5 ml) are desalted with either FastDesalting HR 10/10 or Sephadex G-25 Superfine columns (PharmaciaBiotech, Dubendorf, Switzerland) using 10 mM sodium acetate (pH 5.0) asthe elution buffer. The desalted samples are directly loaded onto a 1.7ml Poros HS/M catio-exchange chromatography column (PerSeptiveBiosystems, Framingham, Mass.) or onto a 1.7-ml Poros HQ/Manion-exchange chromatography column. During both anion-exchange andcation-exchange chromatography, phytase is eluted in pure form by usingan optimized sodium chloride gradient.

Desalted phytases expressed in yeast such as S. cerevisiae or S. pombeare brought to 2 M (NH₄)₂SO₄ after desalting and loaded onto a 1-mlButyl Sepharose 4 Fast Flow hydrophobic interaction chromatographycolumn (Pharmacia Biotech). The enzymes are eluted with a linear 2 to 0M (NH₄)₂SO₄ gradient in 20 mM sodium acetate (pH 5.0). The phytaseseluted in the breakthrough and are concentrated and loaded onto a 120-mlSephacryl S-300 gel permeation chromatography column (PharmaciaBiotech).

For enzymes expressed in Pichia, the enzymes are initially suspendedinto 50 mM Tris-HCl, pH 7, and ammonium sulfate is added to 25% ofsaturation. After the mixture is centrifuged (25,000 g, 20 minutes), thepellet is suspended into 10 mL of 25 mM Tris-HCl, pH 7. The suspensionis dialyzed overnight against the same buffer and loaded onto aDEAE-Sepharose column (Sigma) equilibrated with 25 mM Tris-HCl pH 7.Proteins are eluted with 0.2 M NaCl, 25 mM Tris-HCl, pH 7, after thecolumn is washed with 200 mL of 25 mM Tris-HCl, pH 7. All the collectedfractions are assayed for phytase activity and protein concentration(Lowry et al., 1951). The whole purification is conducted at 4° C., andthe fractions are stored at −20° C.

Estimation of Phytase Activity

Determination of phytase activity, based upon the estimation ofinorganic phosphate released on hydrolysis of phytic acid, can beperformed at 37° C. following the method described by Engelen et al.(2001). One unit of enzyme activity is defined as the amount of enzymethat liberates 1 μmol of inorganic phosphate per minute under assayconditions. For example, phytase activity may be measured by incubating2.0 ml of the enzyme preparation with 4.0 ml of 9.1 mM sodium phytate in250 mM sodium acetate buffer pH 5.5, supplemented with 1 mM CaCl₂ for 60minutes at 37° C. After incubation, the reaction is stopped by adding4.0 ml of a color-stop reagent consisting of equal parts of a 10% (w/v)ammonium molybdate and a 0.235% (w/v) ammonium vanadate stock solution.Phosphate released is measured against a set of phosphate standardsspectrophotometrically at 415 nm. Phytase activity is calculated byinterpolating the A₄₁₅ nm absorbance values obtained for phytasecontaining samples using the generated phosphate standard curve.Alternatively, a phytase activity curve generated by using astandardized phytase reference whose activity is certified by themanufacturer may be used in place of a phosphate standard curve todetermine enzymatic activity. Specific activity can be expressed inunits of enzyme activity per mg of protein.

Alternatively, determination of phytase activities, based on theestimation of inorganic phosphate released on hydrolysis of phytic acid,can be performed at 37EC following the method described by Engelen etal. (1994). One unit of enzyme activity is defined as the amount ofenzyme that liberates 1 μmol of inorganic phosphate per-minute underassay conditions. For example, phytase activity may be measured byincubating 150 ml of the enzyme preparation with 600 ml of 2 mM sodiumphytate in 100 mM Tris HCl buffer pH 7.5, supplemented with 1 mM CaCl₂for 30 minutes at 37° C. After incubation, the reaction is stopped byadding 750 ml of 5% trichloroacetic acid. Phosphate released is measuredagainst phosphate standard spectrophotometrically at 700 nm after adding1500 ml of the color reagent (4 volumes of 1.5% ammonium molybdate in5.5% sulfuric acid and 1 volume of 2.7% ferrous sulfate; Shimizu, 1992).Alternatively, phytase activity is measured in an assay mixturecontaining 0.5% (about 5 mM) phytic acid and 200 mM sodium acetate (pH5.0). After 15 minutes of incubation at 37° C. (or at temperaturesbetween 37 and 90° C.), the reaction is stopped by adding an equalvolume of 15% trichloroacetic acid. The liberated phosphate ions arequantified by mixing 100 ml of the assay mixture with 900 ml of H₂O and1 ml of 0.6 M H₂SO₄-2% ascorbic acid-0.5% ammonium molybdate. After 20minutes of incubation at 50° C., absorbance at 820 nm is measured.Specific activity can be expressed in units of enzyme activity per mg ofprotein.

pH Behavior

For the study of pH behavior, phytase is diluted in 200 mM Na-acetatebuffer, pH 5.5. Substrate solution is prepared in one of the followingbuffers: 200 mM glycine, pH 2.0, 2.5 or 3.0; 200 mM Na-acetate buffer,pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5 and 200 mM Tris-HCl, pH 7.0, 7.5,8.0, 8.5 or 9.0. All buffers are supplemented with 1 mM CaCl₂. Thesubstrate solution contained 10 mM phytic acid from rice (C₆H₆O₂₄Na₁₂;Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

Two milliliters of enzyme preparation are preincubated in a water bathat the assay temperature for 5 minutes, and the enzyme reactions areinitiated by adding 4 ml of the substrate solution. Since the mixingratio slightly alters the pH of the mixture, the pH of the mixture isadjusted to the desired pH before incubation. The mixture is incubatedfor a period, e.g., 60 minutes, at a temperature of 37EC. The incubationis terminated by adding 4 ml of molybdovanadate reagent. The reagent isprepared as described by Engelen et al. (1994). Then the activities ofthe enzyme is determined.

Thermal Behavior

For the determination of the optimum temperature curves, preparation ofenzyme and substrate solutions, as well as their mixing ratio are asdescribed above. However, the pH of the mixtures correspond to thedetermined optimum pH. The mixtures are incubated for a period, e.g., 60minutes, at one or more of the following temperatures: 30, 40, 50, 55,60, 65, 70, 75, 80 and 100EC. The activity is measured on the basis ofinorganic orthophosphate released. For thermostability study in aqueoussolutions, phytases may be preincubated at elevated temperatures. Afterthe preincubation periods, the samples are cooled on ice for 30 minutes.They were reincubated at 37EC and the residual activities of the enzymedetermined.

Thermal stability in aqueous media does not properly reflect stabilityin the feed pelleting process. For an enzyme to be attractive forwidespread application as feed additive, it should be able to withstandtemperature conditions necessary for pre-treatment of feeds. One commonpre-treatment of animal feeds is pelleting. For thermostability study infeed mixtures, a practical diet containing wheat as a major ingredientand fortified with vitamins and minerals may be chosen for pelletingexperiments at different pelleting temperatures. Since wheat contains anappreciable quantity of native phytase activity, the diet is firstpelleted at different temperatures in order to measure the inactivationof the native phytase activity. Heat treatments are varied by modifyingthe steam introduction into the conditioner and temperatures areadjusted in the conditioner (noting that the temperature of the die willincrease for 7 to 10° C. above the temperature in the conditioner).Temperature control in the conditioner is made continuously by a sensorincorporated in the machine. For pelleting, a die with holes of 5 mmdiameter and 15 mm length is used. For calculating the residual activityof added phytases, the native phytase activity at each temperaturetreatment is subtracted from the total activity. The pellets are cooledsubsequently in a batch cooler. Samples of the resulting pellets areanalyzed for the level of phytase activity remaining relative to thatadded to the meal and taking into consideration native phytase activityat each temperature treatment. Most broiler and piglet diets arepelleted at temperatures around 70EC.

Resistance to Protease Inactivation

The resistance of the phytases to protease inactivation may beinvestigated using pepsin from porcine stomach mucosa and pancreatinfrom porcine pancrease. The pepsin, Sigma P7012 (Sigma-Aldrich ChemieGmbH, Steinheim, Germany) contained 2,500 to 3,500 units of activity permg protein and the pancreatin, Sigma P1500, from the same source,contained activity equivalent to the United States Pharmacopeia(U.S.P.). Pepsin is suspended with 0.1 M HCl (pH 2.0) and pancreatin isdispersed in 0.1 M NaHCO₃ (pH 7.0).

For assays with pepsin, 1 ml of a freshly prepared pepsin solutioncontaining 3000 U/ml is mixed with 1 ml of a freshly prepared phytasesolution (0.02 and 0.08 U/2 ml after dilution with buffer at the finalstage of measuring phytase activity) in a test tube. The mixture isincubated for 0 to 45 minutes in a waterbath at 37EC and pH 2.0 (optimumconditions for pepsin activity). After incubation, 1 ml of the solutionis diluted (1:9) with buffer solution (pH 5.5) and thoroughly mixed. Twoml of the solution is incubated with 4 ml phytic acid substrate solutionfor 60 minutes at 40EC and pH 5.5 and phytase activity is determined.For assays with pancreatin, 1 ml of a freshly prepared pancreatinsolution containing 4.81 mg/ml is mixed with 1 ml of phytase solution.The mixture is incubated for 0 and 45 minutes at 40EC and pH 7.0.Dilutions and pH adjustments for phytase activity measurements are thesame as described above.

Alternatively, the purified phytase (2 mg/ml) is incubated withdifferent amounts of pepsin and trypsin following the manufacturerinstructions (Sigma). Pepsin (800 U/mg protein) and trypsin (1500 BAEEunits/mg protein) are dissolved into 10 mM HCl, pH 2 (0.1 mg/mL),respectively. One BAEE unit is defined as 0.001 absorbance change at 253nm per minute at pH 7.6 and 25° C., with BAEE as a substrate. In a finalvolume of 100 mL, 10 mg of purified phytase (0.08 to 0.1 U) is incubatedwith trypsin or pepsin at protease/phytase (w/w) ratios ranging from0.001 to 0.01, at 37° C. for 1 to 120 minutes. The reaction is stoppedon ice and the pH of the mixture was adjusted to 8 for proteinelectrophoresis and phytase activity assay. The digested proteinmixtures were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide orurea-SDS-polyacrylamide gel electrophoresis.

Stability in Digesta Supernatants

Digesta samples are collected from laying hens. The birds are killed bycervical dislocation and their digestive tracts are removed. Digestasamples were collected from crop, stomach (proventriculus), duodenum(pylorus to entrance of bile ducts), jejunum (bile ducts entracts toMeckel's diverticulum), and ileum (Meckel's diverticulum to theileocecal junction). The pH of the digested samples are determined usinga digital pH meter (Ingol Messtechnik AG, Urdorf, Switzerland). The pHreadings of the various segments are 5.02, 2.75, 6.28, 6.63 and 6.98 forcrop, stomach, duodenum, jejunum and ileum, respectively.

The samples are either frozen at −20EC until use or used immediately.Digesta samples are diluted 1:1 in distilled water, mixed thoroughly andcentrifuged a 10,000 g for 10 minutes. Supernatants are recovered andtheir pH values are adjusted to correspond to the initial pH values ofthe different segments of the digestive tract. The recovered digestasupernatants are held in an ice/waterbath until use. For assays, 1 ml ofdigesta supernatants is mixed with 1 ml of enzyme solution and themixture is incubated for 0 and 20 minutes at 40EC. For measuringresidual phytase activity, 1 ml of the solution is diluted (1:9) withbuffer solution (pH 5.5). Two ml of the solution is then mixed with 4 mlof substrate solution and incubated for 60 minutes at 40EC.

EXAMPLE 2 Isolation and Identification of Thermotolerance Phytases

Gene discovery and enzyme optimization, e.g., by combining desirablemutations and/or via DNA shuffling, were employed to identify desiredphytase genes. Thermotolerant phytases were selected and/or optimizedfor desired activity profiles. These include, for example, a highspecific activity (e.g., ≧800 U/mg at pH 4.5 at 37° C. using as asubstrate phytic acid including derivatives thereof, i.e., myoinositolhaving from 1 to 6 phosphate groups, activity at a particulartemperature (e.g., 37° C.), activity at low pH (e.g., a pH optimumbetween 2.5 to 3.5 or less than 4.0 for swine), gastric stability (e.g.,half-life >30 minutes in simulated or actual gastric fluid of poultryand swine), process stability (e.g., half-life ≧5 minutes at 85° C. informulated state, 50% retention of at least activity throughcommercially acceptable pelletization process), lower use rate (e.g.,effective dose of less than 0.5 gram enzyme/ton of feed results inphosphate liberation of more than 75%), and/or substrate specificity(e.g., activity on myo-inositol monophosphate).

A. New Phytases Genes

To identify new phytase genes, a number of different approaches wereused. In one approach, direct cloning of appA genes from 14 different E.coli K-12 strains resulted in 2 new phytase genes, each with 2 aminoacid differences.

To optimize an E. coli phytase (appA) (parent) gene, a saturationmutagenesis was performed in which every codon in the gene was alteredto encode all amino acids. (See, e.g., WO 01/90333, DiversaCorporation). All mutants were tested for residual activity afterheating (70° C.). Sixteen unique clones were identified which hadenhanced thermotolerance relative to the parent gene. Individualmutations were combined in a combinatorial manner, clones prepared foreach combination and the clones were tested for thermal tolerancerelative to wild-type E. coli phytase (FIGS. 1 and 2). The residualactivity profile for clone NOV9X, which has 8 amino acid substitutionsand one silent codon change, after 30 minutes at various temperatures isshown in FIG. 3A and at 100° C. is shown in FIG. 3B. Table 1 summarizesthe properties of various phytases. TABLE 1 Property AppA NatuPhos Nov9xappA-2* SA^(#) 10 1 10 10 Thermal Stability 3% at ND 40% at ND 100° C.100° C.^(#)relative at pH 4.5*2 E. coli gene variants

The gastric stability and thermal tolerance of the NOV9X phytaseexpressed in various host cells is shown in FIG. 4. NOV9X phytase wasproduced in three different microorganisms, E. coli, Pichia pastoris,and Schizosaccharomyces pombe. E. coli does not glycosylate proteins,while Pichia glycosylates proteins to some degree and S. pombe even moreso. An increasing degree of glycosylation appeared to be associated withimproved gastric stability. The data also showed that thermotoleranceincreased with the degree of glycosylation. This effect has not beenobserved with other types of phytases. For example, Wyss et al. (1999)reported that the extent of differential glycosylation had no effect onfungal phytase (A. fumigatus) thermostability. Similarly, Rodriguez etal. (2000) reported no enhanced thermostability of an E. coli phytase(expressed in Pichia pastoris) that was genetically modified to yieldhigher degrees of glycosylation. Thus, the phytase NOV9X has a number ofdesirable properties, e.g., increased thermal tolerance, high specificactivity, and enhanced gastric stability.

With respect to gastric stability and glycosylation, there are only afew comparative studies in the literature with conflicting results. Apaper by Rodriquez et al. (1999) discloses that the parent E. coliphytase gene expressed in Pichia pastoris is very resistant to pepsin,but sensitive to proteolysis by trypsin. Conversely, Natuphos was foundto be resistant to trypsin but sensitive to pepsin.

EXAMPLE 3 Construction and Overexpression of the Nov9X Gene Encoding aPolypeptide with Phytase and Acid Phosphatase Activity in Pichiapastoris

Gene Source and Protein Sequence. A synthetic gene encoding the Nov9Xphytase amino acid sequence (reference to the Diversa patent for theNov9X sequence) was constructed and cloned into the destination cloningvector pPCR-Nov9X. The gene sequence was designed utilizing yeastpreferred codons and supplied in transformed Epicurian Coli XL1-BlueMRF′ cells (Stratagene, La Jolla, Calif.).

Expression Host and Vector. Pichia pastoris pPIC9 expression vector andthe Pichia pastoris GS115 strain were obtained from Invitrogen(Carlsberg, Calif.). The pPIC9 expression vector contains the alcoholoxidase 1 promoter (AOXI) and is methanol inducible. Cloning the Nov9Xgene in frame with the vector's Saccharomyces cerevisiae α-factor prepropeptide secretion signal targets the recombinant protein forextracellular expression.

Construction of Pichia pastoris Transformation Vector. From a plasmidpreparation of pPCR-Nov9X (Qiaprep Spin Miniprep protocol, Qiagen,Valencia, Calif.) the coding region of the target Nov9X gene was excisedby restriction endonuclease digestion using Bgl II and Xba I restrictionenzymes (New England Biolabs, Beverly, Mass.). A typical restrictiondigest was conducted at 37° C. for 60 minutes, followed by heatinactivation for 20 minutes at 65° C. The liberated 1242 base pair DNAfragment was gel purified (QIAquick Gel Extraction Kit, Qiagen,Valencia, Calif.) and used as DNA template for PCR amplification.Synthetic oligonucleotide primers 1 and 2 below (Sigma-Genosys, TheWoodlands, Tex.) and PFU Turbo DNA Polymerase (Stratagene, La Jolla,Calif.) were utilized to amplify the target DNA: Upstream Primer 1: (SEQID NO:2) 5′-gaaggggtat ctctcgagaa aagagaggct caatctgaac cagaattgaagttggaatct Downstream Primer 2: (SEQ ID NO:3) 3′-attattcgcg gccgcctattacaaggaaca ggctgggatt ctA total of 30 cycles using the thermocycling profile listed below wereused to amplify the Nov9X gene:

94° C. for 5 minutes-initial template denaturation

94° C. for 30 seconds-denaturation

61° C. for 30 seconds-annealing

72° C. for 90 seconds-primer extension

Nov9X amplified PCR product (SEQ ID NO:4) was gel purified (QIAquick GelExtraction Kit, Qiagen, Valencia, Calif.) and endonuclease digested withNot I and Xho I (New England Biolabs, Beverly, Mass.). Pichia pastorisexpression vector pPIC9 (Invitrogen, Carlsbad, Calif.) was likewiseprepared by endonuclease digestion with Not I and Xho I and purified bygel extraction. An overnight ligation of endonuclease cut Nov9X PCRproduct with linearized pPIC9 expression vector in the presence of T4DNA ligase at 16° C. (New England Biolabs, Beverly, Mass.) andsubsequent transformation into E. coli Top10F′ competent cells(Invitrogen, Carlsbad, Calif.) produced the Nov9X yeast transformationconstruct. Nov9X/pPIC9 clones containing the gene of interest wereidentified by plasmid DNA restriction mapping with Not I and Xho I. Theintegrity of the Nov9X transformation construct was confirmed by DNAsequence analysis. This cloning strategy produced a construct where theNov9X gene sequence was cloned in frame with the vector's Saccharomycescerevisiae α-factor prepropeptide secretion signal for extra cellularprotein expression.

Preparation of Nov9X/pPIC9DNA for Yeast Transformation. Plasmid DNAcontaining the Nov9X/pPIC9 expression construct was purified from a 50mL culture of E. coli Top10F′cells grown up over night in LB broth thatwas supplemented with 100 μg/mL ampicillin. The isolated plasmid DNA waslinearized by Bgl II endonuclease digestion for 60 minutes at 37° C.Following the digest Bgl II was heat-inactivated by a 20 minuteincubation period at 65° C. Linearized Nov9X/pPIC9 DNA was purified byfirst a phenol and then a phenol-chloroform-isoamyl alcohol extraction.The DNA was precipitated from the aqueous phase of the final extractusing isopropanol, centrifuged, washed with 70% ethanol, and resuspendedin TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0).

Preparation of Pichia pastoris GS115 Competent Cells. Yeast cells wereprepared by streaking the cells onto YPD agarose plates. Following overnight growth at 30° C. a single yeast colony from a YPD agarose platewas transferred to 10 mLs of YPD liquid broth and grown up over night at30° C. From this 10 mL seed culture 100 μL were used to inoculate 500mLs of additional YPD broth. This large scale culture was grown overnight at 30° C. to an optical density of 1.25 when measured at 595 nm.The cells were harvested by centrifugation, resuspended, and treatedwith a series of water and sorbitol washes according to themanufacturer's recommendations (Invitrogen Pichia Expression KitInstruction Manual, version L, pg 59).

Transformation of Nov9X/pPIC9 DNA into Pichia pastoris GS115. Bgl IIrestriction digested Nov9X/pPIC9 plasmid DNA (4.6 μg) was mixed with 80μL of sorbitol treated Pichia pastoris GS 115 cells in a 0.2 cmelectroporation cuvette (Gene Pulser Cuvettes, BioRad, Hercules, Calif.)and incubated on ice for 5 minutes. The electroporation cuvette wasplaced into a BioRad Gene Pulser II instrument and pulsed using settingsof 1.5 kV, 25 μF, and 200Ω. Ice cold sorbitol (1.0 mL) was added to theelectroporation mix which was then plated out onto histidine deficient,minimal media-dextrose (MD) plates. Incubation at 30° C. for up to 3days produced colony growth.

Screening Transformants for Phytase Expression. From the set of primarytransformants plated on MD plates single colonies were inoculated andgrown up over night at 30° C. in 25 mLs of BMGY broth (buffered minimalmedia with glycerol). Genomic DNA was purified from 2 mLs of the 25 mLBMGY liquid cultures using the YeaStar Genomic DNA Purification kit(Zymo Research, Orange, Calif.). Purified genomic DNA along witholigonucleotide primers 1 and 2 listed previously were used in a PCRscreen to identify Pichia pastoris clones harboring our desired phytasegene. Thermocycling conditions listed above were used to test the set ofgenomic clones. Clones generating a PCR fragment of 1281 base pairs werefurther characterized for Nov9X protein expression. The remaining 23 mLsof Pichia culture from clones that tested positive for the Nov9X gene inthe PCR screen were centrifuged at 2000 rpm for 10 minutes, thesupernatant decanted, and the cell pellet resuspended in 10 mLs of BMMY(buffered minimal media with methanol) to induce protein expression.SDS-PAGE analysis of clarified fermentation broth following 24 hours ofincubation at 30° C. identified clones which expressed Nov9X phytase.Functional activity assays measuring the release of inorganic phosphatefrom sodium phytase substrate confirmed phytase expressing cultures thatsecreted functionally active protein.

EXAMPLE 4 Feeding Trials

Mash Feed

FIG. 5 illustrates the effect of dietary inclusion of the NOV9X phytaseon poultry growth performance, represented by feed conversion ratios(FCR). Feed conversion ratio refers to the amount of feed consumeddivided by the net weight gain of the chicken. A lower ratio indicatesthat a chicken gained more weight per unit of feed consumed. A lowerratio indicates that a chicken more efficiently utilized the feed thatwas consumed. Standard poultry diets were used and two inorganicphosphate levels were incorporated into the diets, 0.45% and 0.225%. The0.45% level is commonly used in commercial poultry diets. NOV9X phytase,produced in recombinant P. pastoris was used in this study. Replicatepens of 10 chickens for each diet were grown until 21 days of age, andfinal weights determined by subtracting the weight of the one day oldchicks. Records were kept of the amount of feed consumed by each pen ofchickens, and an average feed consumption was determined. The NOV9Xphytase was formulated by freezing a mixture of liquid enzyme solutionwith a bulking agent, in this instance ground soybean meal, and thenlyophilized. This formulation was added directly to the diets. Natuphoswas used according to the manufacturer's recommendations.

The control diets (with no enzyme supplementation) clearly showed theneed for phosphate supplementation. The low phosphate level gives a FCRof 1.603, while the FCR for the high phosphate control is 1.391. AddingNOV9X phytase at 250, 500, and 1000 U/kg led to an improvement in theFCRs for both the low and high phosphate diets. As more enzyme wasadded, the greater the improvement in the bird growth performance, asindicated by the lower FCR values. Surprisingly, given the markedreduction in phosphate in the low P diet, the 500 U/kg NOV9X lowphosphate diet (FCR of 1.393) performed almost as well as the highphosphate control (FCR 1.391). Moreover, NOV9X phytase performed betterthan Natuphos (at 1040 U/kg), in both the high and low phosphate diets(the manufacturer of Natuphos recommends using a phosphate level of0.45% for the positive control and reducing phosphate by only 0.1% onaddition of 500 U of Natuphos). Thus, the use of the thermotolerantphytase of the invention to supplement feed reduces the levels of addedphosphate needed for enhanced FCR.

Pelleted Feed

A feeding trial similar in design to that described above was performed,except that a pelleted feed was used instead of a mash diet. Feedcomponents were mixed with either the NOV9X phytase enzyme (producedeither in P. pastoris or in S. pombe) or Natuphos and then pelletedusing steam injection for conditioning at 85° C. Replicate pens ofchickens were fed the diets and weight gains were determined in 42 dayold chickens.

A control diet with no added phytase contained 0.45% phosphate. Allother diets (with Natuphos or NOV9X at 100, 300, and 900 units/kg)contained 0.225% phosphate. The weight gain data is shown in FIG. 6.These data show that the NOV9X phytase survived the pelletizationprocess and resulted in improved performance of the chickens thatconsumed those diets. Weight gains significantly improved relative tothe no enzyme control and are approximately equal to the positivecontrol (the 0.45% high phosphate diet with no enzyme). These data alsoconfirmed the superior performance of the NOV9X phytase relative toNatuphos with respect to thermostability.

EXAMPLE 5 Feeding Trials

Seven further trials were conducted, some to 21 days of age and othersto 42 days of age. In each trial NOV9X was dosed at various levels andin most cases compared against Natuphos. Animal performance data fromthese 7 and the 2 trials described above were then entered into adatasheet and statistically analysed using a stepwise linear regressionapproach to determine which of the 35× variables (diet, enzyme andmanagement variables) examined described the variation in the datasetbest. Significant models were described for both gain and FCR and areexpressed graphically in FIGS. 7 and 8. Enzyme source (i.e., Natuphos orNOV9X) proved to be a significant determinant of the gain and fcrvariation measured. NOV9X therefore proved to be superior to Natuphos onaverage, over the 9 trials entered in the dataset, in terms of both gainand fcr. Such a multi-factorial or meta-analyis approach is morereliable in determining relative efficacies of products since unduereliance on one trial is avoided.

Taken collectively, the data in this example indicate that the NOV9Xphytase was significantly more effective at liberating organic phosphatethat is present in the soybean and corn portions of the feed. When NOV9Xphytase is used, it is clear animal performance can be maintained withthe addition of less inorganic phosphate than is necessary in thepresence of natuphos. This suggests that there will be a net reductionon phosphorus in the manure with use of NOV9X compared with Natuphoswhen diets are formulated to take advantage of capabilities of eachproduct.

Additionally, these results indicate that novel compound animal feedswith low inorganic phosphate levels can be used to efficiently producefarm animals in a geographically intense manner while significantlyreducing the environmental release of phosphate in the excreta of theanimal. This means that farms producing these animals will have less ofan environmental impact.

REFERENCES

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All cited publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1-29. (canceled)
 30. An animal feed composition comprising a thermotolerant phytase which retains at least 40% activity after 30 minutes at 60° C. and has a specific activity of greater than 200 U/mg at pH 4.5 and 37° C., wherein the thermotolerant phytase is encoded by a nucleic acid molecule comprising the nucleic acid sequence depicted in SEQ ID NO:4 encompassed by the restriction enzyme sites XhoI and NotI or a conservative variant thereof, or wherein the thermotolerant phytase comprises the polypeptide sequence of SEQ ID NO:1 or a conservative variant thereof.
 31. The animal feed composition of claim 30, wherein the phytase has a specific activity of greater than 400 U/mg at pH 4.5 and 37° C.
 32. The animal feed composition of claim 30, wherein the phytase has a specific activity of greater than 600 U/mg at pH 4.5 and 37° C.
 33. The animal feed composition of claim 30, wherein the phytase has a specific activity of greater than 800 U/mg at pH 4.5 and 37° C.
 34. The animal feed composition of claim 30, wherein the thermotolerant phytase has a half life in simulated gastric fluid of greater than 25 minutes at a pH greater than 2.0 and less than 4.0.
 35. An enzyme feed additive comprising a thermotolerant phytase which retains at least 40% activity after 30 minutes at 60° C. and has a specific activity of greater than 200 U/mg at pH 4.5 and 37° C., wherein the thermotolerant phytase is encoded by a nucleic acid molecule comprising the nucleic acid sequence depicted in SEQ ID NO:4 encompassed by the restriction enzyme sites XhoI and NotI or a conservative variant thereof, or wherein the thermotolerant phytase comprises the polypeptide sequence of SEQ ID NO:1 or a conservative variant thereof.
 36. The enzyme feed additive of claim 35, wherein the thermotolerant phytase has a specific activity of greater than 400 U/mg at pH 4.5 and 37° C.
 37. The enzyme feed additive of claim 35, wherein the thermotolerant phytase has a specific activity of greater than 600 U/mg at pH 4.5 and 37° C.
 38. The enzyme feed additive of claim 35, wherein the thermotolerant phytase has a specific activity of greater than 800 U/mg at pH 4.5 and 37° C.
 39. The enzyme feed additive of claim 35, wherein the thermotolerant phytase has a half life in simulated gastric fluid of greater than 25 minutes at a pH greater than 2.0 and less than 4.0. 40-72. (canceled)
 73. The animal feed composition of claim 30, wherein the thermotolerant phytase is isolated.
 74. The enzyme feed additive of claim 35, wherein the thermotolerant phytase is isolated. 